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Synthetic nucleic acid molecule compositions and methods of preparation

Active Publication Date: 2006-03-30
PROMEGA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0015] The nucleotide substitutions in the synthetic nucleic acid sequence may be influenced by many factors such as, for example, the desire to have an increased number of nucleotide substitutions such as those resulting in a silent nucleotide substitution (encodes the same amino acid) and / or decreased number of regulatory sequences. Under some circumstances (e.g., to permit removal of a transcription factor binding site) it may be desirable to replace a non-preferred codon with a codon other than a preferred codon or a codon other than the preferred codon in order to decrease the number of regulatory sequences.
[0021] Also provided is a synthetic (including a further synthetic) nucleotide sequence prepared by the methods of the invention, e.g., a further synthetic nucleotide sequence in which introduced regulatory sequences or restriction endonuclease recognition sequences are optionally removed. Thus, the method of the invention may be employed to alter the codon usage frequency and / or decrease the number of regulatory sequences in any open reading frame or to decrease the number of regulatory sequences in any nucleic acid sequence, e.g., a noncoding sequence. Preferably, the codon usage frequency in a synthetic nucleotide sequence which encodes a selectable or screenable polypeptide is altered to reflect that of the host organism desired for expression of that nucleotide sequence while also decreasing the number of potential regulatory sequences relative to the parent nucleic acid molecule.

Problems solved by technology

TFBS searches were limited to vertebrate TF binding sites.

Method used

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  • Synthetic nucleic acid molecule compositions and methods of preparation
  • Synthetic nucleic acid molecule compositions and methods of preparation
  • Synthetic nucleic acid molecule compositions and methods of preparation

Examples

Experimental program
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Effect test

example 1

Synthetic Click Beetle (RD and GR) Luciferase Nucleic Acid Molecules

[0107] LucPp / YG is a wild-type click beetle luciferase that emits yellow-green luminescence (Wood, 1989). A mutant of LucPplYG named YG#81-6G01 was envisioned. YG#81-6G01 lacks a peroxisome targeting signal, has a lower KM for luciferin and ATP, has increased signal stability and increased temperature stability when compared to the wild type (PCT / WO9914336). YG #81-6G01 was mutated to emit green luminescence by changing Ala at position 224 to Val (A224V is a green-shifting mutation), or to emit red luminescence by simultaneously introducing the amino acid substitutions A224H, S247H, N3461, and H348Q (red-shifting mutation set) (PCT / WO9518853) Using YG #81-6G01 as a parent gene, two synthetic gene sequences were designed. One codes for a luciferase emitting green luminescence (GR) and one for a luciferase emitting red luminescence (RD). Both genes were designed to 1) have optimized codon usage for expression in mamm...

example 2

Synthetic Renilla Luciferase Nucleic Acid Molecule

[0224] The synthetic Renilla luciferase genes prepared include 1) an introduced Kozak sequence, 2) codon usage optimized for mammalian (human) expression, 3) a reduction or elimination of unwanted restriction sites, 4) removal of prokaryotic regulatory sites (ribosome binding site and TATA box), 5) removal of splice sites and poly(A) sites, and 6) a reduction or elimination of mammalian transcriptional factor binding sequences.

[0225] The process of computer-assisted design of synthetic Renilla luciferase genes by iterative rounds of codon optimization and removal of transcription factor binding sites and other regulatory sites as well as restriction sites can be described in three steps: [0226] 1. Using the wild type Renilla luciferase gene as the parent gene, codon usage was optimized, one amino acid was changed (T→A) to generate a Kozak consensus sequence, and undesired restriction sites were eliminated thereby creating synthetic...

example 3

[0271] Synthetic Firefly Luciferase Genes

[0272] The luc+gene (U.S. Pat. No. 5,670,356) was optimized using two approaches. In the first approach (Strategy A), regulatory sequences such as codons were optimized and consensus transcription factor binding sites (TFBS) were removed (see Example 4, although different versions of programs and databases were used). The sequences obtained for the first approach include hluc+ver2AF 1 through hluc+ver2AF8 (designations with an “F” indicate the construct included flanking sequences). hluc+ver2AF1 is codon-optimized, hluc+ver2AF2 is a sequence obtained after a first round of removal of identified undesired sequences including transcription factor binding sites, hluc+ver2AF3 was obtained after a second round of removal of identified undesired sequences including transcription factor binding sites, hluc+ver2AF4 was obtained after a third round of removal of identified undesired sequences including transcription factor binding sites, hluc+ver2AF5...

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Abstract

A method to prepare synthetic nucleic acid molecules having reduced inappropriate or unintended transcriptional characteristics when expressed in a particular host cell.

Description

BACKGROUND [0001] Transcription, the synthesis of an RNA molecule from a sequence of DNA is the first step in gene expression. Sequences which regulate DNA transcription include promoter sequences, polyadenylation signals, transcription factor binding sites and enhancer elements. A promoter is a DNA sequence capable of specific initiation of transcription and consists of three general regions. The core promoter is the sequence where the RNA polymerase and its cofactors bind to the DNA. Immediately upstream of the core promoter is the proximal promoter which contains several transcription factor binding sites that are responsible for the assembly of an activation complex that in turn recruits the polymerase complex. The distal promoter, located further upstream of the proximal promoter also contains transcription factor binding sites. Transcription termination and polyadenylation, like transcription initiation, are site specific and encoded by defined sequences. Enhancers are regulat...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C07H21/04
CPCC07K14/43595C12N15/70C12N15/65C07K2319/00
Inventor WOOD, KEITHWOOD, MONIKAALMOND, BRIANPAGUIO, AILEENFAN, FRANK
Owner PROMEGA
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